Endothelial Rap1 in the control of heart function

内皮 Rap1 控制心脏功能

基本信息

批准号：
10366802
负责人：
Magdalena Chrzanowska
金额：
$ 73.88万
依托单位：
VERSITI WISCONSIN, INC.
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-01-19 至 2025-12-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10366802
关键词：
Affect American Attention Automobile Driving Biological Availability Blood Vessels Blood flow Cardiac Cardiac Myocytes Cardiac development Cardiovascular Diseases Cell Communication Cell physiology Cessation of life Coculture Techniques Collaborations Communication Coronary Coronary Vessels Data Defect Development Disease Electrophysiology (science)Endothelial Cells Endothelium Evaluation Functional disorder Future Goals Growth Health Heart Heart Contractilities Heart failure Homeostasis Human Impairment In Vitro Incidence Ion Channel Knockout Mice Knowledge Lead Maintenance Measurement Mediating Metabolism Molecular Monomeric GTP-Binding Proteins Mus Nitric Oxide Paracrine Communication Pathway interactions Periodicity Persons Phenotype Physiology Population Production Protein Isoforms Regulation Research Role STIM1 gene Signal Pathway Signal Transduction Smooth Muscle Myocytes Testing Therapeutic Intervention Tissues Vascular Permeabilities Vasodilation adverse outcome endothelial dysfunction endothelial repair heart function heart preservation in vivo mortality novel pandemic disease paracrine patch clamp postnatal preservation prevent release factor repaired response shear stress therapy design transcriptomics

项目摘要

Cardiovascular disease (CVD) is a global pandemic with over 26 million people affected worldwide. Critical for regulation of heart oxygenation and metabolism is the cross-talk between heart endothelial cells (ECs) and cardiomyocytes (CMs) and smooth muscle cells (SMCs). This cross-talk, mediated by locally acting, bioactive substances released by cardiac ECs (paracrine function), in particular nitric oxide (NO), controls blood flow and vascular permeability, as well as CMs' growth, contractility and rhythmicity. However, the mechanisms underlying the functional interaction between cardiac ECs and CMs and SMCs are still poorly understood. Our pioneering studies on endothelial functions of the small GTPase (Ras Association Proximate) Rap1 highlight its role as novel regulator of vascular homeostasis. Rap1 is critically required for nitric oxide (NO) production and bioavailability, as tissue-specific deletion of both Rap1 isoforms (Rap1A and Rap1B) leads to severe endothelial dysfunction. Emerging data from our collaboration strongly suggest that the two Rap1 isoforms in both coronary (vascular) and heart microcapillary (cardiac) ECs may be essential to preserving normal contractile function of the heart. Our data demonstrate that EC-specific deletion of Rap1 leads to decreased cardiac contractility and impending heart failure. Mechanistically, our preliminary data strongly suggest that, via discrete yet complementary mechanisms, two Rap1 isoforms are essential for endothelial Ca2+handling and endothelial function (NO production). The goal of this proposal is to examine the role of the two Rap1 isoforms in coronary and cardiac ECs required for maintenance of cardiac contractile function. We hypothesize that Rap1-dependent EC functions form the nexus for EC-SMC and EC- CM communication required for normal cardiac function. Conversely, Rap1 deficiency-driven EC dysfunction (impaired NO release, Ca2+ overload) is the common culprit in EC–SMC and EC–CM miscommunication that leads to heart failure. To test this hypothesis, we will: (1) Determine how Rap1 controls Ca2+ homeostasis in ECs; we will utilize patch clamp electrophysiology and Ca2+ measurements in vitro to examine the effect of Rap1 deficiency on Ca2+ influx channels. We will examine the effect of impaired Ca2+ homeostasis in Rap1A KO ECs on cellular processes controlling paracrine function. (2) Examine a novel signaling pathway involving CalDAG-GEFIII-mediated Rap1B activation in NO release. Ex vivo, we will test the effect of Rap1 signaling and ion channel inhibition on mouse and human coronary vessel dilation, to determine the influence of EC Rap1A and Rap1B in the control of coronary vessel blood flow. (3) Examine vascular and cardiac function in EC-Rap1 knockout mice ex vivo and paracrine function in EC-CM co-culture in vitro to determine how cardiac EC Rap1 isoforms control heart contractile function. Proposed studies will uncover novel, previously unexpected mechanisms governing heart endothelium and may lead to a new direction in restoring cardiac function by controlling Rap1 signaling in endothelium.

心血管疾病（CVD）是全球大流行，全球有超过2600万人受到影响。至关重要心脏氧合和代谢的调节是心脏内皮细胞（EC）和心肌细胞（CMS）和平滑肌细胞（SMC）。这个串扰，由本地作用，生物活性介导心脏ECS（旁分泌功能）释放的物质，特别是一氧化氮（NO），控制血流以及血管通透性以及CMS的生长，收缩性和节奏性。但是，机制心脏ECS与CMS和SMC之间的功能相互作用的基本相互作用仍然知之甚少。我们对小GTPase（RAS关联）RAP1的内皮功能的开创性研究强调了其作为血管稳态的新型调节剂的作用。一氧化氮至关重要（NO）需要RAP1 生产和生物利用度，作为两种RAP1同工型（RAP1A和RAP1B）的组织特异性缺失导致严重的内皮功能障碍。我们合作的新兴数据强烈表明这两个RAP1 冠状动脉（血管）和心脏微毛细血管（心脏）EC中的同工型可能对保存至关重要心脏的正常收缩功能。我们的数据表明，EC特定的RAP1的删除会导致精致的心脏收缩性和即将到来心脏衰竭。从机械上讲，我们的初步数据强烈表明，通过离散而完整机理，两个RAP1同工型对于内皮CA2+处理和内皮功能至关重要（否生产）。该提案的目的是检查两种RAP1同工型在冠状动脉和心脏中的作用维持心脏收缩功能所需的EC。我们假设Rap1依赖性EC 功能构成了正常心脏功能所需的EC-SMC和EC-CM通信的联系。相反，RAP1缺乏驱动的EC功能障碍（没有释放受损，Ca2+过载）是常见的 EC – SMC和EC -CM沟通沟渠中导致心力衰竭的罪魁祸首。为了检验这一假设，我们将：（1）确定RAP1如何控制EC中的Ca2+稳态；我们将利用斑块夹电生理学和 CA2+在体外的测量值检查RAP1缺乏对Ca2+影响通道的影响。我们将检查 RAP1A KO EC中Ca2+稳态受损的影响对控制旁分泌功能的细胞过程。（2）检查一种新的信号通路涉及caldag-gefiii介导的RAP1B激活，无释放。前任体内，我们将测试RAP1信号传导和离子通道抑制对小鼠和人冠状动脉的影响扩张，以确定EC RAP1A和RAP1B在控制冠状血液流动中的影响。（3）检查EC-rap1基因敲除小鼠的血管和心脏功能在EC-CM中的旁分泌功能和旁分泌功能体外共培养，以确定心脏EC RAP1同工型如何控制心脏收缩功能。拟议的研究将发现有关心脏内皮的新颖，以前出乎意料的机制，通过控制内皮中的RAP1信号传导来恢复心脏功能的新方向。